Microbiological outcomes from different periodontal maintenance interventions : a systematic review

Abstract: This study aimed to investigate the differences in the subgingival microbiological outcomes between periodontal patients submitted to a supragingival control (SPG) regimen as compared to subgingival scaling and root planing performed combined with supragingival debridement (SPG + SBG) intervention during the periodontal maintenance period (PMP). A systematic literature search using electronic databases (MEDLINE and EMBASE) was conducted looking for articles published up to August 2016 and independent of language. Two independent reviewers performed the study selection, quality assessment and data collection. Only human randomized or non-randomized clinical trials with at least 6-months-follow-up after periodontal treatment and presenting subgingival microbiological outcomes related to SPG and/or SPG+SBG therapies were included. Search strategy found 2,250 titles. Among these, 148 (after title analysis) and 39 (after abstract analysis) papers were considered to be relevant. Finally, 19 studies were selected after full-text analysis. No article had a direct comparison between the therapies. Five SPG and 14 SPG+SBG studies presented experimental groups with these respective regimens and were descriptively analyzed while most of the results were only presented graphically. The results showed that both SPG and SPG+SBG protocols of PMP determined stability in the microbiological results along time. Nevertheless, new studies comparing these interventions in PMP are needed, especially if the limitations herein discussed could be better controlled.


Introduction
The literature has clearly shown that the maintenance of periodontal results after an adequate therapy is delivered depends on the quality of the supragingival plaque control (SPG). 1,2,3,4,5However, during the past 20 years the subgingival scaling and root planing (SBG) has been associated to that control and this association (SPG+SBG) been recognized as the choice therapy during periodontal maintenance period (PMP). 6,7,8Nevertheless, studies comparing this combination to other protocols were not available until recently when a clinical study 9 and two systematic reviews 10,11 suggested that the SPG biofilm control alone might be the choice during PMP.The Patricia Daniela Melchiors ANGST (a) Amanda Finger STADLER (b) Rui Vicente OPPERMANN (c) Sabrina Carvalho GOMES (c) (a) Universidade Federal de Pelotas -UFPEL, Dental School, Department of Semiology and Clinic, Pelotas, RS, Brazil.
(b) Augusta University, The Dental College of Georgia, Department of Periodontics, Augusta, GA, United States of America.
(c) Universidade Federal do Rio Grande do Sul -UFRGS, Dental School, Department of Conservative Dentistry, Porto Alegre, RS, Brazil.
authors showed no clinical outcomes benefits from the addition of the subgingival scaling (SPG + SBG).Microbiological composition of the subgingival area after therapy as well is an issue of discussion.It is suggested that microbiological differences between biofilms may explain part of the failures observed along PMP period.The impact that the subgingival instrumentation has over the amount and composition of the biofilm has been shown. 12owever, not only the subgingival intervention but also the supragingival control modifies the quality and quantity of the subgingival biofilm. 13,14,15Besides, evidences support the biofilm as the main source of bacteria to the subgingival area. 16So, in theory, both interventions, singly, would be able to maintain a microbiota compatible with periodontal stability and health along years.Taking this plausibility in mind, this systematic review aimed at investigating how does the subgingival microbiota respond to the SPG in comparison with the SPG+SBG as maintenance protocols.

Methodology
This systematic review was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic and Meta-Analysis (PRISMA) Statement 17 and used the reference manager Mendeley (version Desktop 1.16.1-OSX-Universal;Elsevier Inc., New York, USA).Neither a protocol nor a systematic review registration was considered.

Focused question
In periodontal patients submitted to PMP, is there a difference in the subgingival microbiological outcomes from the SPG regimen as compared to the SPG+SBG intervention?

Search strategy
Studies were identified by electronic databases (MEDLINE via PubMed, and EMBASE) searching for studies published up to August 2016.No language restrictions were applied.The following MesH terms were used in different arrangements (Appendix 1-A; 1-B): randomized clinical trial; randomized controlled trial; clinical trial; longitudinal study; prospective study; supportive periodontal care; periodontal maintenance, and microbiology.
A manual search of the reference list from narrative and systematic reviews studies, as well as the bibliographies of the included studies, was performed.

Study selection
Two independent reviewers (PDMA and AFS) nominated the articles that initially met the search criteria, based on their titles.After agreement on selected articles, the same 2 examiners read the abstracts independently.Once the agreement was again accessed, the articles were elected for full-text reading.The final inclusion of articles was done after discussion and verbal agreement between the 2 examiners.A third examiner (SCG) reviewed any disagreement between the examiners during all phases.
The selection of the articles was performed according to: a.

Quality assessment
Method of randomization, blinding of examiners, rate of patient losses during the follow-up, and the protocol and periodontal status information were evaluated to access the quality of the studies selected based on an adaptation of the Jadad scale. 18,19ach item was scored: 0: not shown or absent information; 1: incomplete or unclear information, or 2: complete/adequate information.The achievement of a sum score of at least 4 was necessary to the study to be included (Appendix 2).A total score of 4 or 5 corresponded to an adequate quality, whereas a final score of 6 to 8 corresponded to a good quality.

Data extraction
Data were extracted considering: 1) the methodology of the studies and 2) the microbiological finds.

Statistical analysis
Inter-examiner agreement for studies selection based on titles and abstracts was calculated using the Kappa coefficient.
As a consequence of the great discrepancy how results were presented by the authors, assessment of statistical heterogeneity or a meta-analysis could not be conducted.

Searching results
The electronic searches resulted in 3,563 (MEDLINE) plus 879 (EMBASE) and the manual in 19 titles.After extracting duplicate citations, 2,250 potential articles remained to be screened (dated from 1968 on).From these, 148 after title and 39 after abstract analysis were considered to full-text evaluation.This evaluation resulted in the inclusion of 19 articles (Figure 1; Appendix 3).Kappa values for inter-examiner agreement were 0.71 and 0.79 for titles and abstract analyses, respectively.
Table 1 shows the results of the quality assessment of the included studies.All studies presented, at least, adequate quality (Appendix 2).
Among the included studies, none reported microbiological outcomes from direct comparisons between SPG versus SPG+SBG interventions.Five studies reported only SPG results, while 14 studies reported SPG+SBG.The characteristics of the included studies are shown in Table 1.

SPG results
Table 2 reports the microbiological data regarding the 5 SPG studies.The results from two studies 20,21 were interpreted by visual analysis from graphics and in accordance with the author's conclusions/statements.
Conventional Polimerase Chain Reaction (PCR): Chondros et al. 20 showed stability or even a decrease in the mean levels of T. forsythia, A. actinomycetemcomitans, P. gingivalis, T. denticola and F. nucleatum between 3 and 6 months of PMP, while P. intermedia presented a slight (non significant) increase.
Checkerboa rd DNA-DNA hybridi zat ion: Colombo et al. 21showed that three months past the PMP beginning, the levels of all target species were maintained as stable, although the prevalence of A. actinomycetemcomitans and F. nucleatum experienced an increase.
Culture: Quirynem et al. 22 reported significant reductions in detection frequencies for P. gingivalis and P. intermedia in the first months of PMP, followed by stability over time.Rosling et al. 23 observed a small decrease on the total viable counts (TVC) between PMP baseline (15 x 10 6 ) and PMP-36-month (12 x 10 6 ) examination.Nevertheless, there was a significant reduction in the number of patients positive for P. gingivalis and A. actinomycetemcomitans, and a stability of those values for P. intermedia.Finally, in the study by Westfelt et al. 24 the mean TVC (10 6 ) were reduced between PMP baseline (20.1 ± 22 x 10 6 ) and 36-month (5.4 ± 11.9 x 10 6 ) examinations, and a decrease in the mean percentage of P. gingivalis (14.7 ± 25.4 to 0.4 ± 0.3); and P. intermedia (9.6 ± 11.6 to 3.5 ± 5.0) was also observed.Moreover, the number of patients positive for A. actinomycetemcomitans and P. intermedia was relatively stable over time, whereas a decrease was observed for P. gingivalis (4 patients to 1).

SPG + SBG results
The microbiological results of the 14 SPG+SBG studies are also presented in Table 2.Among these studies, only four reported the microbiological outcomes by numeric results on tables. 25,26,27,28eal-time PCR: Kolbe et al. 27 showed a non-significant trend to decrease the quantities (amounts: log 10 ± SEM) of P. gingivalis (1.6 ± 1.4 to 1.0 ± 1.4) and T. forsythia (4.9 ± 3.6 to 4.7 ± 3.8) from PMP baseline to 6 months, respectively, and stability for A. actinomycetemcomitans (2.7 ± 3.1 to 2.7 ± 2.5).There were no differences over time in the percentage of sites harboring T. forsythia and A. actinomycetemcomitans, whereas a significant decrease in the frequency of P. gingivalis was observed.In the same way, Müller et al. 28 reported an overall stability on microbiological outcomes since the number of positive sites with counts at > 1,000 and > 100,000 cells/ml were not significantly different before and after 12 months of PMP, for any of the target microorganisms."Significant reductions in the prevalence and levels were observed for P. gingivalis, T. forsythia and A. actinomycetemcomitans.Although this last species reduced significantly in counts over time, its frequency increased to baseline values at 9 mo post-therapy.""Species of Prevotella showed a modest decrease in prevalence, however, their levels were markedly reduced." "the most striking changes in prevalence and levels of the majority of the microorganisms occurred during the first 3mo after SRP, although several species still presented lower levels at 9mo when compared with baseline values." "these results indicate that periodic maintenance visits are needed to keep the pathogenic species at lowered levels.""The number of aerobic as well as anaerobic species around SR (…) remained nearly unchanged for the placebo group (small treatment effect with a 0.3 log reduction).""For the MR the changes were comparable with similar intra-as well as inter-product variations."Rosling et al. 1997  "Mean counts of 13 of 40 and 8 of 40 target species changed significantly over time in the T and C groups, respectively.In particular, species in the green complex showed significant reductions over time in C group, whereas species in the green and orange complexes were significantly reduced over time in the T group." Cortelli et al. "P.gingivalis, T. forsythia and T.denticola decreased in prevalence and levels up to the 6mo visit and remained at these lower levels at 9-and 12mo".
"proportion of the total DNA probe count that T. forsythia, P. gingivalis comprised was significantly decreased at 12mo".
"[T. forsythia and P. gingivalis] declined in prevalence until 6mo and showed a slight increase at 9-and 12mo post therapy whereas proportions continued to decrease.The decline in proportion of these species paralleled the decrease in PPD." "most profound reduction occurred during the first 3mo post SRP although these species were still reduced significantly at 12mo when compared with pre-treatment levels.Thus, maintenance scaling appeared to be important in maintaining the initial post therapy decreases in selected species for prolonged periods of time."

Ehmke et al. 2005 31
Data expressed as % of patients colonized.C: control group; T: test group "both therapies had only a limited influence on the prevalence of the majority of assessed periodontal pathogens.With the exception of A. actinomycetemcomitans no long-term eradication of pathogens was registered over the entire study period." "No additional differences in detection frequencies of P. gingivalis were found between T and C group patients over the study period." "No significant differences were found between T and C group concerning the prevalence of T. forsythia, Treponema ssp., and P. intermedia." "In 5 T-group patients and 1 C-group patient, A. actinomycetemcomitans was suppressed over the 24mo study period.In the remaining patients, A. actinomycetemcomitans was temporarily suppressed or persisted."Gunsolley et al. 1994 26   Data expressed as mean levels (mean proportion of positive sites).
"There was a significant decrease in total counts for (…) subgingival plaque samples in the subjects using the manual brush and a significant decrease in subgingival counts for the P group.The majority of subjects in both groups showed a decrease in total counts from baseline to 6 mo." "All taxa examined were reduced in prevalence for both brushing groups.Only A. actinomycetemcomitans increased in prevalence in both groups.""prevalence of the red complex species, T. forsythia, P. gingivalis, and T. denticola was markedly decreased for most subjects.""The major finding was the effect of supragingival plaque removal on the composition of the subgingival microbiota.""Total bacterial scores showed an overall significant difference by time for the C group (p = 0.002) and a borderline difference for the T group (p = 0.05).
The difference between groups was not significant.Comparable values of bacterial counts were recorded at different time points for the two treatments.""In the C group, a significant reduction in bacterial counts was observed from baseline to 6 mo (p = 0.008) and to 12 mo (p = 0.003), whereas the reduction between 6 mo and 12 mo was not significant.The reduction in bacterial counts for the T group from baseline to 6-and 12 mo was close to significant, whereas the reduction between 6-and 12 mo was not significant." "For both treatments, the prevalence of P. gingivalis decreased significantly from baseline to 6mo (control: p = 0.016; test: p = 0.039), and within the T group, a significant reduction was observed from baseline to 12 mo (p = 0.016).At 12 mo, P. gingivalis was totally eradicated in the C group, whereas one patient in the T group harbored P. gingivalis." "prevalence of T. forsythia decreased significantly from baseline to 6 mo within the T group (p = 0.021) and the pathogen was not detected at 12 mo.In the C group, the reduction from baseline to 6-and 12 mo was not significant." "significant reduction in prevalence of A. actinomycetemcomitans from baseline to 6 mo was only achieved for the C group (p = 0.008).From 6 to 12 mo, a rebound was observed for both treatments."

Continuation
Listgarten et al. 1989 34 Data expressed as mean proportion of bacterial morphotypes.C: control group; T: test group.
"proportions of coccoid cells were not significantly different between the C and T groups.Proportions were highest during the first 6 mo, dropping sharply at the 1-year examination.(…) the proportions increased slightly with time, but never reached baseline levels.""% of motile rods means did not differ significantly between groups, nor did they differ between examinations." "mean proportions of spirochetes throughout the study was similar for the C and T group.The proportions decreased from baseline through the first 18mo.After stabilizing for the next 18mo, they began to increase again.""percentages of "other" bacterial morphotypes for both treatment groups increased from the baseline and 6mo examinations to the 1-year and subsequent examinations.There were no differences between the proportions of other bacteria for the C and T groups.""patients in the T group fared as well as the C patients both clinically and microbiologically." .D-group: control group.
"There was a general pattern of increasing prevalence of bacterial pathogens with increasing time in both groups.From 6 mo on, there were significantly more patients in the D-group with high counts of P. gingivalis and T. forsythia than in the M-group.No difference was found for A. actinomycetemcomitans and P. intermedia." "At 12 mo, 68% [of individuals] in the D-group had one or more sites with at least two species of the Red Complex at counts > 10 5 . The corresponding percentage of the sites amounted 35.9%, respectively." "prevalence of P. gingivalis, T. forsythia, A. actinomycetemcomitans, P. intermedia remained at levels ≤ 10 "The detection frequencies of the studied microorganisms at >1,000 and >100,000 cells/ml were not significantly different before and after 12 months.However, at the final examination the frequency of sites with counts of A. actinomycetemcomitans >1000 cells/ml was lower in the test compared to the control group, and no sample contained >100,000 cells/ml, compared to two in the control group."): Day 0: 37.7 x 10 5 ; 36 mo: 20.9 x 10 5 .
"Both clinical groups showed statistically significant reductions in the total mass of subgingival biofilm as measured by the total DNA probe counts"

Ximenez-Fyvie et al. 2000 38
Data expressed as mean total and individual species counts (x 10 5 ).
"Mean total counts and mean counts of individual species (34/40) decreased after completion of the professional cleaning phase and continued to decrease even though the subjects returned to self-performed plaque control after the 3mo monitoring visit." "The major effect was seen at 3mo, immediately after completion of the professional cleaning phase, for P. gingivalis and T. forsythia.Mean counts of A. actinomycetemcomitans were decreased at 3mo but continued to decline between 3-and 6mo." "reduction in mean counts observed was due in part to a decrease in prevalence (% of sites colonized), but more to a reduction in the % of sites exhibiting high counts of the test species.5 species were significantly decreased.P. gingivalis was found not only at significantly fewer sites but in significantly lower numbers." "Therapy employed was able to establish a host compatible microbiota for a prolonged period of time."Conventional PCR: Cortelli et al. 25 observed no significant differences between PMP time-points in the presence of target pathogens.Irrespective of theses observations, reductions on mean bacteria values for A. actinomycetemcomitans (3.70 to 3.30) and P. intermedia (4.20 to 3.20), between 3 and 12 months of PMP, respectively, were significant.Ehmke et al. 31 showed that the results related to the prevalence of A. actinomycetemcomitans and P. gingivalis presented a general stability along PMP.However, an increase in the frequency of P. intermedia and T. forsythia could be observed.Interestingly, these values were somewhat similar to those observed at pre-treatment examination.
Checkerboard DNA-DNA hybridization: studies performed by Bogren et al. 29 , Haffajee et al. 32 , Teles et al. 37 and Ximenez-Fyvie et al. 38 showed that the mean levels/counts (x10 5 ; SEM) of the target species remained constant 29 or even decreased 32,37,38 during PMP.The results from Cugini et al. 30 showed an overall stability in prevalence and counts of all target bacteria, especially for T. forsythia and P. gingivalis, with some fluctuations between study time-points, although the increases never reached the pre-treatment values.Krohn-Dale et al. 33 , in turn, observed a significant decrease in the total bacterial scores and number of positive patients for the target bacteria between PMP-day 0 and 6-month examination, followed by stability or even a light decreasing until the end of the study.On the other hand, there was a rebound for A. actinomycetemcomitans at the PMP-12-month exam.In opposition, McColl et al. 35 showed a general pattern of increase in the number of individuals or sites (counts > 10 5 ) positive to the target bacteria species over PMP, with the final numbers always greater than the pre-treatment values.
Dark field microscopy: Listgarten et al. 34 reported that the mean proportions of different bacterial morph-types were maintained stable during PMP.However, the mean values for the so-called "other bacteria morph-types" increased from PMP-day 0 and 6-month examinations to 1-year and subsequent examinations.Finally, Murray et al. 36 showed a significant shift in the microbiota for the experimental group that experienced high percentage of obligate anaerobes, motile rods, and spirochetes at the beginning of PMP.After 12 months, the microbiota showed similarity to that described as associated to oral health status.F. nucleatum (log) total colony-forming unit (CFU) values were relatively unchanged from day 0 to 6-months, but had significantly decrease at 12-months.

Discussion
The present study sought to investigate subgingival microbiological outcomes during the PMP when supragingival biofilm control alone or its combination with the subgingival one were delivered.Although no randomized clinical trial aiming to answer this question was available, the results from studies with SPG or SPG + SBG experimental groups showed that both interventions were able to propitiate stability on the microbiological results over time.
There was an expressive variation in PMP baseline definition in the studies once a clear distinction between treatment end and PMP commencement phase were absent in most of them.Because of that, the 90 th day after therapy was considered the PMP baseline to data collection. 1,39,40If the 90 th day data was not available, the nearest exam was considered.This was applied to 3 out of 5 SPG studies, and in 8 out of 13 SPG+SBG studies.Besides, most of the studies included presented short-term evaluations of no longer than 12 months of PMP.It is stated that this is a short period to observe great changes in periodontal status over time, and that the major challenges for a breakdown or recurrence of the disease occur later in the maintenance phase. 2,8,37,41,42erein, microbiological findings in studies with more than 12 months showed in general stability along subsequent years. 23,24,29,34,37he methodology for microbiological identification performed by most studies were qualitative or semi-quantitative analyses, when it is accepted that molecular identification and absolute quantification are the preferred techniques. 43,44Irrespective, the overall results showed constancy of the subgingival microbiological outcomes during PMP related to both interventions, even thought changes in bacteria numbers could be observed within experimental time-points.These fluctuations were particularly observed in the SPG+SBG studies reporting the percentage of positive patients/sites (e.g., prevalence) by the use of Checkerboard DNA-DNA hybridization 30,33,35 or a non-quantitative method (dark field microscopy 34 and conventional PCR 31 ).Interestingly some of the fluctuations observed indicated eradication of some bacterial type.However, as it is known that the eradication of oral pathogens residents is not possible and even an undesirable goal, 14,16 this so called eradication should be interpreted with caution.The absence or disappearance of bacterial specie might be related to limitations of the methods. 43,44he main limitation of the preset study is that it was not possible to perform a statistical analysis.Results reported only graphically and differences between studies methods, such as different periods of evaluation and the microbiological method applied, made the meta-analyses impossible.However, the pattern of microbiological response was very consistent between studies, irrespective of the applied therapy: SPG or SPG + SBG.In addition, in accordance with the literature, 12,13,14 it was observed a close relationship between these microbiological and the clinical outcomes reported, somewhat reinforcing the similarity of response between the PMP investigated protocols.Notwithstanding, similar findings have already been reported by Heasman et al. 10 in a systematic review of clinical data from different studies others than those included in the present review.

Conclusion
The importance of the present results relies on the fact that microbiological outcomes during PMP are, in part, considered cause of failure of the periodontal stability over time.Consequently, this assumption reinforces the need to access the subgingival area as to reduce the chances to lose attachment levels along time.However, it seems that both SPG and SPG + SBG regimens during PMP determined stability in the microbiological results along time.Nevertheless, new studies comparing this outcome are still needed aiming to improve the quality of the future researches on this field.

Table 1 .
Characteristics of the included studies.

Table 2 .
Microbiological outcomes of the included studies.Study Microbiological results or remarkable findings as stated by the authors of the studies SPG studies Chondros et al. 2009 20 Data expressed as mean bacterial levels.No information was reported regarding intra-group comparisons in the control group."bothtreatments resulted in significant reductions in [mean level of] T. denticola, F. nucleatum, (…).The only differences between the two treatments were statistically significant reductions of (…) E. corrodens and Capnocytophaga sp. at 6mo after treatment with PDT." 5in the majority of patients and sites in both groups."Müller et al. 2014 28 Data expressed as numbers of positive sites with counts > 1000 and > 100,000 cells/ml (respectively) [n = 50].Day 0: A. actinomycetemcomitans: 7 and 0; T. forsythia: 39 and 15; P. gingivalis: 37 and 12; T. denticola: 42 and 27; P. intermedia: 14 and 6; 12mo: A. actinomycetemcomitans: 10 and 2; T. forsythia: 35 and 19; P. gingivalis: 34 and 10; T. denticola: 38 and 17; P. intermedia: 11 and 4.
Appendix 2. Quality assessment of the included studies.Appendix 3. Excluded full-text analyzed studies.